Limits...
Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.

Gemmecker S, Schaub P, Koschmieder J, Brausemann A, Drepper F, Rodriguez-Franco M, Ghisla S, Warscheid B, Einsle O, Beyer P - PLoS ONE (2015)

Bottom Line: Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase.This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins.This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.

ABSTRACT
Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

No MeSH data available.


Related in: MedlinePlus

Spectral properties of PDS and roles of quinones and of norflurazon.A Trace; UV-VIS spectra of PDS-His6 that was purified in the presence of norflurazon. The low absorbance in the 450 nm region and the high one in the 310 nm range indicates the presence of enzyme-bound reduced flavin (spectra recorded after 2h at RT in an oxygen atmosphere). Trace b, spectra of the same sample upon heat-denaturation of the protein (5 min 100°C). Trace c; spectrum of PDS upon addition of 200 μM DPQ (recorded after 20 min). B, Dependence of the rate of ζ-carotene formation on the Eo’and structure of the quinone electron acceptor. The structures of the naphtoquinones (☐) and benzoquinones (○) used are given in S3 File. Decyl-plastoquinone (100 mV) is highlighted (●).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492965&req=5

pone.0131717.g008: Spectral properties of PDS and roles of quinones and of norflurazon.A Trace; UV-VIS spectra of PDS-His6 that was purified in the presence of norflurazon. The low absorbance in the 450 nm region and the high one in the 310 nm range indicates the presence of enzyme-bound reduced flavin (spectra recorded after 2h at RT in an oxygen atmosphere). Trace b, spectra of the same sample upon heat-denaturation of the protein (5 min 100°C). Trace c; spectrum of PDS upon addition of 200 μM DPQ (recorded after 20 min). B, Dependence of the rate of ζ-carotene formation on the Eo’and structure of the quinone electron acceptor. The structures of the naphtoquinones (☐) and benzoquinones (○) used are given in S3 File. Decyl-plastoquinone (100 mV) is highlighted (●).

Mentions: The presence of the herbicide norflurazon (50 μM) in all buffers used during purification led—in addition to the stabilizing effect discussed above—to the isolation of the reduced, colorless PDS-His6 protein. The reduced form was stable for over 2 h in ambient oxygen atmosphere (Fig 8A, trace a). Heat denaturation led to rapid oxidation upon FAD-release and appearance of yellow color (trace b). We deduce from this observation that the PDS-His6-bound FAD is reduced by an unknown donor in E.coli and that the reduced colorless form is stabilized by association with norflurazon. Interestingly, rapid reoxidation can also be achieved with excess decyl-plastoquinone (DPQ; trace c), indicating that the quinone and norflurazon compete at the FAD site.


Phytoene Desaturase from Oryza sativa: Oligomeric Assembly, Membrane Association and Preliminary 3D-Analysis.

Gemmecker S, Schaub P, Koschmieder J, Brausemann A, Drepper F, Rodriguez-Franco M, Ghisla S, Warscheid B, Einsle O, Beyer P - PLoS ONE (2015)

Spectral properties of PDS and roles of quinones and of norflurazon.A Trace; UV-VIS spectra of PDS-His6 that was purified in the presence of norflurazon. The low absorbance in the 450 nm region and the high one in the 310 nm range indicates the presence of enzyme-bound reduced flavin (spectra recorded after 2h at RT in an oxygen atmosphere). Trace b, spectra of the same sample upon heat-denaturation of the protein (5 min 100°C). Trace c; spectrum of PDS upon addition of 200 μM DPQ (recorded after 20 min). B, Dependence of the rate of ζ-carotene formation on the Eo’and structure of the quinone electron acceptor. The structures of the naphtoquinones (☐) and benzoquinones (○) used are given in S3 File. Decyl-plastoquinone (100 mV) is highlighted (●).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492965&req=5

pone.0131717.g008: Spectral properties of PDS and roles of quinones and of norflurazon.A Trace; UV-VIS spectra of PDS-His6 that was purified in the presence of norflurazon. The low absorbance in the 450 nm region and the high one in the 310 nm range indicates the presence of enzyme-bound reduced flavin (spectra recorded after 2h at RT in an oxygen atmosphere). Trace b, spectra of the same sample upon heat-denaturation of the protein (5 min 100°C). Trace c; spectrum of PDS upon addition of 200 μM DPQ (recorded after 20 min). B, Dependence of the rate of ζ-carotene formation on the Eo’and structure of the quinone electron acceptor. The structures of the naphtoquinones (☐) and benzoquinones (○) used are given in S3 File. Decyl-plastoquinone (100 mV) is highlighted (●).
Mentions: The presence of the herbicide norflurazon (50 μM) in all buffers used during purification led—in addition to the stabilizing effect discussed above—to the isolation of the reduced, colorless PDS-His6 protein. The reduced form was stable for over 2 h in ambient oxygen atmosphere (Fig 8A, trace a). Heat denaturation led to rapid oxidation upon FAD-release and appearance of yellow color (trace b). We deduce from this observation that the PDS-His6-bound FAD is reduced by an unknown donor in E.coli and that the reduced colorless form is stabilized by association with norflurazon. Interestingly, rapid reoxidation can also be achieved with excess decyl-plastoquinone (DPQ; trace c), indicating that the quinone and norflurazon compete at the FAD site.

Bottom Line: Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase.This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins.This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, Cell Biology, University of Freiburg, Freiburg, Germany.

ABSTRACT
Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

No MeSH data available.


Related in: MedlinePlus